Effect of proteinase-activated receptor 2 activating peptide, 2-furoyl-LIGRLO-amide, on murine mesenteric endothelial cell Ca²⁺-events

Hennessey, John C. (2015) Effect of proteinase-activated receptor 2 activating peptide, 2-furoyl-LIGRLO-amide, on murine mesenteric endothelial cell Ca²⁺-events. Masters thesis, Memorial University of Newfoundland.

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Abstract

Proteinase-activated receptor 2 (PAR2)-mediated elevations of cytosolic Ca²⁺, induced by PAR2 agonist 2-furoyl-LIGRLO-amide (2fly), have been demonstrated in cell culture experiments. The properties of individual PAR2-mediated Ca²⁺-events in endothelial cells (EC) have yet to be described, and the role that these Ca²⁺-events play in the preservation of PAR2-mediated vascular reactivity in endothelial dysfunction is not understood. Male C57BL/6J (PAR2-WT) and PAR2 knockout (PAR2-KO) mice were implanted with sub-cutaneous micro-osmotic pumps containing either saline (control) or angiotensin II (endothelial dysfunction). Doses of angiotensin II were 1.5 mg/kg/day, control pumps were filled with 100 μl of saline. Imaged under spinning disk confocal microscopy, Fluo-4 loaded Ang II-treated ECs exhibited 21% and 23% attenuation in the density of ACh-evoked muscarinic (M₃)-mediated, Ca²⁺-events in PAR2-WT and PAR2-KO, respectively. 2fly-mediated Ca²⁺-events were unaffected by Ang II treatment. The density of Ca²⁺-release sites were reduced by IP3R inhibition using xestospongin-C and TRPV channel inhibition using ruthenium red. This study identified two populations of non-propagating Ca²⁺-events in murine mesenteric ECs: small peripheral and large-repeating transients. The study concluded that PAR2 Ca²⁺-signaling is preserved in the presence of endothelial dysfunction. Immunocytochemistry experiments of fixed and permeabilized ECs, on a line-scan confocal microscope, revealed that the distribution of eNOS was significantly attenuated with Ang II treatment. This reduction of eNOS in Ang II-treated ECs may contribute to the attenuation of resistance vessel relaxation in endothelial dysfunction.

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